1. FIELD OF THE INVENTION
The present invention relates to apparatus for biological sample preparation and, more particularly, to apparatus for automatically mixing hematological sample material with chemical reagents in a rapid, gentle, specific, selectable, controllable timed sequence.
2. DESCRIPTION OF THE PRIOR ART
Flow cytometry provides investigators with data on physical parameters of particles, for example, human cells. One of the cell types of greatest interest is the human leukocyte (white cell). Human leukocytes can be classified into different classes and subclasses. These classifications in part are made by comparing data from physical measurements made on these cells. These measurements include cell volume and granularity. Immunological classification can be performed by using monoclonal antibodies tagged with fluorescent compounds. Physical measurements can be used to classify white cells into three major groups, for example, lymphocytes, monocytes, and granulocytes. Immunofluorescent measurements can be used to classify the lymphocytes into subclasses, for example, T-cells, B-cells, etc. Immunofluorescence also can be used to further classify the T-cells into at least two categories, for example, cytotoxic cells, and suppressor cells, etc. The ability to classify human white cells has provided investigators with valuable diagnostic information. However, in order for investigators to analyze leukocytes with flow cytometry, the sample must be free of other cell types, such as red blood cells. Therefore, sample preparation is an important step required to insure rapid reproducible results.
The current methods of leukocyte preparation for immunofluorescence include whole blood lysis techniques and density gradient separation techniques. The major lytic techniques use either ammonium chloride or detergents to lyse red cells. These lytic techniques require the immunological staining to take place before red cell lysis. The current techniques fix or require fixation of the leukocytes immediately after lysis, in order to maintain sample stability. In current commercially available lytic procedures, the sample must be washed at least once sometime during the procedure. Lytic techniques have the advantage of requiring only 100 .mu.l of whole blood per sample to perform each test. In density gradient separation, the lymphocytes are separated from most of the other blood cells on the basis of their buoyant density. This technique usually requires between 3 and 6 ml of whole blood in order to perform several stainings. It also differs from lytic techniques because the immunofluorescent staining is carried out after the lymphocytes are removed from the red blood cells. Density gradient separation also requires that the sample be washed at least one during the procedure. One advantage of density gradient separations is that they do not require immediate fixation after the lymphocytes are stained.
All the above mentioned preparation techniques are manual in nature. In lytic techniques, the operator monitors the lyse times and the sample must be visually monitored for complete red cell lysis. In density gradient methods, the operator must remove the specific density layer containing the lymphocytes without collecting any other cells in other layers. In addition, the operator must centrifuge then wash away unwanted material. Obviously, this activity risks biohazard contamination and cell viability. These manual techniques contribute to variability in flow cytometric data.